Progress and Challenges in Understanding the Biology, Diversity, and Biogeography of Cenococcum Geophilum

Chapter 14 Progress and Challenges in Understanding the Biology, Diversity, and Biogeography of Cenococcum geophilum

Keisuke Obase, Greg W. Douhan, Yosuke Matsuda, and Matthew E. Smith

14.1 Introduction

Cenococcum geophilum Fr. (Fries 1825) (syn. C. graniforme Ferd. and Wing.; Ferdinandsen and Winge 1925) was described as an anamorphic, melanized fungus characterized by the production of jet-black, hard, spherical sclerotia in forest soils (Massicotte et al. 1992). Since Linhell (1942) found that C. geophilum forms ectomycorrhizal associations with woody plants, the combination of the sclerotia, the ectomycorrhizal nutritional mode, and the distinct morphology of the ectomycorrhizas (Agerer and Gronbach 1988; Ingleby et al. 1990; Agerer and Rambold 2004–2016) have been accepted as crucial characters to identify C. geophilum (Fig. 14.1). C. geophilum was one of the ﬁrst ectomycorrhizal fungi to be studied in great detail. In a seminal work, Trappe (1964) showed that C. geophilum has an extremely wide host range and forms ectomycorrhizas with gymnosperms (such as species of Pinaceae) and angiosperms (such as species of Fagaceae, Betulaceae,

K. Obase (*) Department of Mushroom Science and Forest Microbiology, Forestry and Forest Products Research Institute, 1 Matsunosato, Tsukuba, Ibaraki 305-8687, Japan e-mail: [email protected] G.W. Douhan Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521, USA Cooperative Extension Advisor, Tulare Co., University of California, Tulare, CA 93274, USA Y. Matsuda Laboratory of Forest Mycology, Graduate School of Bioresources, Mie University, Kurimamachiya 1577, Tsu, Mie 514-8507, Japan M.E. Smith Department of Plant Pathology, University of Florida, 2523 Fiﬁeld Hall, Gainesville, FL 32611-0680, USA

Fig. 14.1 Cenococcum geophilum.(a) An ectomycorrhizal root of Betula ermanii that has been colonized by C. geophilum. (b) Fresh sclerotia of C. geophilum after they were extracted from forest soils. (c) Hyphae extending from a C. geophilum sclerotium as it begins to grow in axenic culture on an agar plate. Bars ¼ 0.5 mm and Salicaceae). C. geophilum is also widely distributed in boreal, temperate, and subtropical regions where compatible host plants grow and is also often a major component of ectomycorrhizal fungal communities (Trappe 1962, 1964; Dahlberg et al. 1997; LoBuglio 1999; Horton and Bruns 1998). It is no exaggeration to say that C. geophilum is ubiquitous on roots of ectomycorrhizal woody plants in most natural and second-growth forests, although it appears that C. geophilum may be absent or rare on ectomycorrhizal trees in some tropical biomes (Tedersoo et al. 2010; Smith et al. 2011). At the global scale, C. geophilum is considered one of the most ubiquitous ectomycorrhizal fungi in forest soils and on woody plant roots. Despite the fact that C. geophilum is common across many habitats on multiple continents, no sexual or asexual spores have ever been convincingly recorded for this fungus and we do not actually know how the fungus spreads in nature. Fernańdez-Toirań and A´ gueda (2007) recorded a cleistothecium that they considered to be a fruitbody of C. geophilum. However, the identity of this cleistothecium was not confirmed based on direct physical connection between mycorrhizas and fruitbody or using molecular tools, and therefore remains open to interpretation and doubt. The available evidence suggests that C. geophilum disperses clonally via sclerotia and hyphal growth between root tips and should therefore be limited to short-distance dispersal. Although the short-distance exploration type of mycorrhizas (Agerer 2001), limited hyphal growth, and sclerotia formation in C. geophilum suggest the likelihood of short-distance dispersal, these morphological observations contrast with the fact that C. geophilum is abundant and ubiquitous in many forests. These contradictory observations suggest that population biology studies using 14 Progress and Challenges in Understanding the Biology, Diversity, and... 301 molecular tools are needed to elucidate the biology of C. geophilum, explain how it is dispersed, and determine if it undergoes sexual recombination. The inability to mate strains of C. geophilum in the lab has previously hampered accurate identification of the fungus and limited our understanding of genetic variation and genetic spatial patterns in this fungus. However, starting in the 1990s advances in molecular techniques clarified the phylogeny, ecology, and systematics of C. geophilum. Phylogenetic studies have recently shown that C. geophilum is a member of the Gloniaceae (Dothideomycetes, Ascomycota; Spatafora et al. 2012). Molecular approaches can also convincingly identify samples of C. geophilum (Matsuda et al. 2015) to genotype level and this approach will be critical to understand the dispersal mode of this fungus as well as elucidating the population structure at various spatial scales from soil cores to regions to continents (e.g., Wu et al. 2005; Douhan et al. 2007a; Matsuda et al. 2015). Evidence from population studies and from phylogenetic analyses all suggested that there is some cryptic recombination process that occurs in C. geophilum (LoBuglio and Taylor 2002; Douhan et al. 2007b; Bourne et al. 2014; Matsuda et al. 2015). Moreover, a recent study found a sex-related gene (MAT1-1-1) and genes encoding pheromone response proteins that are involved in the formation of fruiting bodies in the genome of a C. geophilum isolate (Peter et al. 2016). This suggests that C. geophilum likely is able to form fruiting bodies and reproduce sexually. Simultaneously, however, molecular data have clarified several critical issues for understanding the spatial genetic distribution and population biology of C. geophilum. First, C. geophilum is monophyletic but either an extremely heterogeneous species or (more likely) a species complex (LoBuglio et al. 1991). A series of studies indicated high local and global genetic diversity within C. geophilum and the presence of several cryptic lineages that are likely distinct species (Douhan and Rizzo 2005; Douhan et al. 2007a; Matsuda et al. 2015; Obase et al. 2016a). Because all of these lineages look essentially identical in the morphology of their sclerotia, root tips, and axenic cultures, there is an accidental risk of including phylogenetically distant isolates in population genetic analyses. As pointed out by Douhan and Rizzo (2005), there have also been cases where melanized, sclerotia-forming fungi from outside of the monophyletic C. geophilum lineage have accidentally been included in population studies and may have generated spurious results. Another factor to consider is that genetic diversity is occasionally high even within individual soil cores due to co-occurrence of distinct lineages at a small spatial scale. The co-occurrence of multiple distantly related lineages within individual soil cores means that the results of population genetic and phylogenetic diversity studies will be directly related to the amount of sampling effort that is expended. An additional but related issue is that the C. geophilum genotype pools detected from ectomycorrhizas may be systematically different from the pool of isolates obtained from sclerotia (Obase et al., unpublished). In addition to those issues, there are relatively few studies that have examined the diversity of C. geophilum outside of the USA, Europe, and Japan so our global view of this group of fungi is still limited to certain regions. The populations and lineages of C. geophilum in Africa, South America, Australasia, and most of central Asia remain almost completely unknown. 302 K. Obase et al.

In this chapter we revisit the host range and global distribution of C. geophilum, which has not been compiled in a review since the overview provided by Trappe (1964). We will also discuss the challenges for understanding the biogeography of C. geophilum in light of the high number of cryptic species, the co-existence of multiple lineages at small spatial scales, and the unknown aspects of the lifecycle of C. geophilum. We discuss the implications of the most recent in-depth studies that revealed spatial genetic structure of one lineage of C. geophilum at larger geographical scales in Japanese pine forests. Finally, we discuss future research directions that will be needed to understand the spatial genetic structure of a common but enigmatic ectomycorrhizal fungus, C. geophilum.

14.2 Host Range and Distribution

The global host range and distribution of C. geophilum was summarized by Trappe (1964) but a large number of mycorrhizal studies and an excellent review (LoBuglio 1999) have been published since that landmark paper. As a host for C. geophilum, Trappe (1964) listed 129 species/variations/hybrids in Pinaceae (Abies, Larix, Picea, Pinus, Pseudotsuga, Tsuga), Betulaceae (Alnus, Betula, Carpinus, Corylus), Ericaceae (Arctostaphylos), Fagaceae (Castanea, Castanopsis, Fagus, Lithocarpus, Nothofagus, Quercus), Myrtaceae (Eucalyptus), Rosaceae (Cercocarpus, Chamaebatia, Rosa, Sorbus), Salicaceae (Populus, Salix) and Tiliaceae (Tilia). In addition to these original reports, C. geophilum has been reported on a large number of temperate or arctic-alpine woody host genera: Adenostoma; Allen et al. (1999), Arbutus; Molina and Trappe (1982), Cassiope;Va¨re et al. (1997), Cathaya;Vaarioetal.(2006),Cedrus;Bakshi et al. (1968), Chosenia; Hashimoto and Higuchi (2003), Dryas; Haselwandter and Read (1980), Ostryopsis; Bai et al. (2003), and Photinia; Grand (1971). C. geophilum has also been reported from several genera of tropical and subtropical woody hosts in Dipterocarpaceae [Dipterocarpus;Phosrietal.(2012),Dryobalanops, Hopea, Parashorea; Brearley et al. (2003), Shorea; Brearley et al. (2007), Tristaniopsis; Alexander and Hogberg€ (1986)] as well as in Coccoloba uvifera (Seńe et al. 2015). In addition to the genera of trees and shrubs mentioned above, C. geophilum has also been shown to form ectomycorrhizas with herbaceous species in Cistaceae (Helianthemum, Hudsonia, Lechia) (Malloch and Thorn 1985; Dickie et al. 2004), Cyperaceae (Kobresia) and Polygonaceae (Polygonum) (Massicotte et al. 1998; Mühlmann et al. 2008) as well as with mycoheterotrophic plants in the genera Hemitomes, Pleuricospora,andPterospora (Castellano and Trappe 1985). As we can deduce from the exceedingly wide host range, C. geophilum is widely distributed in boreal, temperate, and subtropical regions (Trappe 1962, 1964; Dahlberg et al. 1997; LoBuglio 1999; Horton and Bruns 1998). Most ectomycorrhizal studies in tropical regions have reported that C. geophilum is absent or infrequent in ectomycorrhizal fungal communities (Die´dhiou et al. 2010; Tedersoo et al. 2010; Smith et al. 2011; Corrales et al. 2016). However, several recent studies have documented C. geophilum from tropical biomes, suggesting that novel and 14 Progress and Challenges in Understanding the Biology, Diversity, and... 303 undiscovered host associations are likely to be found from locations where the ectomycorrhizal fungal communities were not sufficiently surveyed (Morris et al. 2008; Phosri et al. 2012; Dokmai et al. 2015; Seńe et al. 2015). C. geophilum has been found in contrasting climatic regions, including arctic (Fujiyoshi et al. 2011) and subarctic (Hrynkiewicz et al. 2009) to tropical (Phosri et al. 2012) and subtropical regions (Trappe 1962; Obase et al. 2016a). C. geophilum is also found across wide elevational gradients from coastal forests near sea level (Matsuda et al. 2009a, b; Obase et al. 2009, 2011; Seńe et al. 2015) to alpine habitats at or near treeline (Hasselquist et al. 2005). C. geophilum is often dominant in ectomycorrhizal fungal communities that are exposed to high drought stress, including pine forests on sand dunes (Matsuda et al. 2009a, b; Obase et al. 2009, 2011) and seasonally dry woodlands and savannahs (Smith et al. 2007). Also, C. geophilum is found in serpentine soils that are known to have phytotoxic levels of Mg and/or Ni (Panaccione et al. 2001; Moser et al. 2009). Factors involving the wide ecological niche have not been fully investigated but one possibility is that the high levels of melanin in fungal cell walls may contribute to tolerance of environmental stress such as drought (Fernandez and Koide 2013) and toxicity from heavy metals. This result has been previously shown in melanized pathogenic fungi (Go´mez and Nosanchuk 2003). Recently, a genomic study of C. geophilum uncov- ered patterns that may partly explain the wide ecological niche of the fungus. C. geophilum has a larger genome (ca. 178 Mb) compared with other Dothideomycetes owing to the high content of transposable elements (Peter et al. 2016). Transposable elements are correlated with the plasticity and adaptability of fungi to their environment (e.g., Casacuberta and Gonza´lez 2013). Last, sclerotia of C. geophilum are excellent resting structures that may remain active for several years (Trappe 1962; Miller et al. 1994). These structures act as a spore bank and readily colonize host plant roots in response to disturbance like other disturbance- adapted ectomycorrhizal fungi such as Rhizopogon spp. Indeed, C. geophilum sclerotia are the most resistant structures of ectomycorrhizal fungi and they can survive long-lasting drought treatments and readily survive soil heating of 45–60 C (Izzo et al. 2006; Glassman et al. 2015; Miyamoto and Nara 2016).

14.3 Phylogenetic Diversity in the C. geophilum Species Complex

The phylogenetic position and the closest relatives of C. geophilum have been unknown until recently, because no sexual and asexual spores were recorded. Based on similar morphological characteristics of sclerotia, anatomical features in hyphae and the ability to form ectomycorrhizas with woody plants, C. geophilum was historically hypothesized to be an anamorphic stage of Elaphomyces (Eurotiales, Ascomycota) (Ferdinandsen and Winge 1925; Trappe 1971). Co-occurrence of C. geophilum and Elaphomyces spp. in several forests also supported this idea. 304 K. Obase et al.

However, LoBuglio et al. tested the hypothesis using rDNA hybridization (LoBuglio et al. 1991) and phylogenetic analysis of the 18S rDNA (LoBuglio et al. 1996). They found that C. geophilum is genetically distinct from Elaphomyces spp. and is not a close relative. More recently, Spatafora et al. (2012) analyzed the phylogenetic relationships of C. geophilum with other members of Dothideomycetes (Ascomycota) based on five loci. They found that C. geophilum is closely related to the genus Glonium and is an isolated ectomycorrhizal lineage not closely related to any other known mycorrhizal fungi. The ecology of Glonium is not well understood, but species in this genus are likely non-mycorrhizal saprobes that inhabit soil or decaying wood (Kantvilas and Coppins 1997). Interestingly, species of Glonium form darkly pigmented, carbonaceous ascomata (modified hysterothecia—Boehm et al. 2009). It is not known if Glonium species form sclerotia in soils. A BLAST search based on the ITS sequence of Glonium stellatum deposited in MycoCosm (Grigoriev et al. 2014; http://jgi.doe.gov/fungi) revealed that similar sequences have been detected from ericaceous plant roots and suggest that some members of the genus may be able to colonize roots. Obase et al. (2016a) recently discovered a fungus that was isolated from surface-sterilized Cenococcum- like sclerotia from soil but this fungus was not resolved within the C. geophilum lineage. This species was described as Pseudocenococcum floridanum K. Obase, G.W. Douhan, Y. Matsuda and M.E. Smith and is genetically closer to C. geophilum than to species of Glonium. P. floridanum is morphologically similar to C. geophilum but grows faster in culture and did not form ectomycorrhizas with pine and oak seedlings. The fungus is likely a saprobe and the closest known relative of C. geophilum. The discovery of these close relatives of C. geophilum (Glonium and Pseudocenococcum) suggests that the ancestor of C. geophilum was morphologically similar to P. floridanum and C. geophilum, grew in forest soil and formed sclerotia but was probably not ectomycorrhizal. Due to the lack of closely related ectomycorrhizal fungi and the distinct morphological characteristics of ectomycorrhizas, C. geophilum is regarded as a unique ectomycorrhizal fungus that can be identified reliably to the ‘species’ level based solely on morphological characteristics of ectomycorrhizal roots. However, previous studies have often found diverse cultural and physiological characteristics among C. geophilum isolates (LoBuglio 1999), indicating genetic diversity and/or the presence of cryptic species in C. geophilum. LoBuglio et al. (1991) were the first to document high genetic variation among C. geophilum isolates from geographically divergent locations in the USA and Europe. This was the first evidence to indicate that C. geophilum was either an extremely heterogeneous species or a species complex. Even though the ITS region is rather conserved within C. geophilum (Shinohara et al. 1999), high genetic diversity was nonetheless detected in a series of studies using a variety of molecular biology methods that sampled at various spatial scales from forest stands to regions to continents (Panaccione et al. 2001; Portugal et al. 2001; Jany et al. 2002; Douhan and Rizzo 2005; Wu et al. 2005; Chen et al. 2007; Gonçalves et al. 2007; Bahram et al. 2011; Spatafora et al. 2012; Matsuda et al. 2015; Obase et al. 2016a). For example, Douhan and Rizzo (2005) found three phylogenetically distinct lineages within 14 Progress and Challenges in Understanding the Biology, Diversity, and... 305

C. geophilum populations from one oak stand in California based on glyceralde- hyde 3-phosphate dehydrogenase (GAPDH), the ITS region, the mitochondrial SSU (mit SSU), and an intron in the 18S rDNA. Douhan et al. (2007b) further examined genetic variation using the GAPDH gene sequence by adding isolates of C. geophilum from Europe but found that phylogeographic inference was obscured and that the backbone nodes of this larger phylogeny had poor bootstrap support. Interestingly, however, isolates from different continental origins were often intermingled in the phylogenetic tree. Obase et al. (2016a) revisited the phylogenetic diversity of C. geophilum at an intercontinental scale by using new data from Florida (USA) with existing data from Douhan et al. (2007b) and Japan (Matsuda et al. 2015) based on two loci (ITS and GAPDH). The combination of the two loci resolved six well-supported lineages and some of them included isolates from different geographical regions, as shown in Douhan et al. (2007b). Re-analysis of the phylogeny of a smaller subset of isolates with more genes (ITS, GAPDH, SSU, LSU, TEF, RPB1, and RPB2) conﬁrmed the uniqueness of the six cryptic lineages but also resolved some higher-level relationships among them (e.g. clades 1, 2 and 4 are clustered together with a 87% bootstrap support—Fig. 14.2). Although the ITS region is not the ideal locus for delineating lineages within C. geophilum sensu lato (Obase et al. 2016a), this DNA region can nonetheless be used to identify additional phylogenetic diversity within C. geophilum (Bahram et al. 2011) by using the massive sequence data that are deposited in the UNITE database (Ko˜ljalg et al. 2013). Three-hundred-forty-four ITS sequences from putative C. geophilum were available by searching with the query “Cenococcum” in the UNITE database (https://unite.ut.ee/, accessed October 2016). These sequences originate from various geographic regions, including North and South America, Europe and Asia. They can be divided into 12 groups based on 97% sequence similarity cutoff. Most of the ITS sequences were uniﬁed into one putatively monophyletic group that includes sequences from different geographical regions (n ¼ 318). However, a few sequences from North America (Canada and USA), Asian countries (China, Thailand, Pakistan) and Sweden were resolved into distinct groups. In addition, several unique C. geophilum ITS groups that are delineated by 97% sequence similarity were detected in forests across various geographical regions (e.g., Ge et al. 2012; Huang et al. 2014). Although it is possible that these distinct groups of ITS sequences could be chimeras generated during PCR or artefacts of low read quality, cloning, or sequencing, it is also possible that these could be unique, undescribed species that are more distantly related to C. geophilum sensu lato. We expect that extensive sampling of ectomycorrhizal roots and sclerotia from different geographical regions and with phylogenetically unique host plants is likely to yield a large number of unique lineages of C. geophilum that match these unique ITS sequences, much like we found in our intensive studies in Florida, USA (Obase et al. 2016a). All available evidence suggests that C. geophilum is a species complex. Therefore, it is extremely important not to include phylogenetically-distinct lineages of C. geophilum together in analyses of spatial genetic structure and population biology. When unrelated C. geophilum lineages are inadvertently mixed together for these types 306 K. Obase et al.

Fig. 14.2 The optimum phylogenetic tree of C. geophilum based on maximum likelihood analysis of seven concatenated loci (ITS, SSU, LSU, TEF, RPB1, RPB2 and GAPDH). The placement of each lineage is highlighted and clades 1–6 are named according to Obase et al. (2016a). Inset in the upper left corner shows the known global distribution of each lineage based on multi-gene data (although vast areas of the globe have not been sampled). Isolates of P. floridanum are included as outgroups. CA: California, USA, FL: Florida, USA, FR; France, NL: Netherland, OR: Oregon, USA, SP: Spain, SW: Switzerland of analyses it is inevitable that we will infer erroneous spatial genetic patterns. Multi- locus phylogenetic analysis, even based on only two loci (Obase et al. 2016a), is an important option for determining the phylogenetic affinities of C. geophilum samples and then selecting adequate data for the analysis of spatial genetic structure. The procedure is also useful for excluding unrelated dematiaceous fungi that can be accidentally obtained from sclerotia of C. geophilum as contaminants (Douhan et al. 2007a; Obase et al. 2016b) or non-ectomycorrhizal fungi that are related to C. geophilum, such as P. floridanum (Obase et al. 2016a). For example, Jany et al. (2002) documented high genotypic diversity of C. geophilum at the scale of individual soil cores (10 10 10 cm) and a pattern of isolation by distance in five beech forests in northeastern France across approximately 250 km, using PCR/RFLP of the ITS region and sequence characterized amplified region (SCAR1) with the HinfI endonu- clease. However, a subsequent study found that some samples in the Jany et al. (2002) study were likely not C. geophilum based on LSU-rDNA or GAPDH gene sequences 14 Progress and Challenges in Understanding the Biology, Diversity, and... 307 and that their sampling probably included isolates from multiple lineages of C. geophilum (Douhan et al. 2007b). This makes interpretations regarding population genetic issues difficult.

14.4 High Genetic Diversity at Small Spatial Scales

We have a quite limited view of the genetic diversity of ectomycorrhizal fungi at a fine spatial scale (Douhan et al. 2011; Chap. 2). Many fruiting ectomycorrhizal fungi are known to form genets with extending hyphae from several centimeters to meters in soils (Douhan et al. 2011). One genotype often dominates within the soil samples collected from the central area of the genet (e.g., Tricholoma matsutake; Lian et al. 2006; Tuber melanosporum; Murat et al. 2013). In the case of C. geophilum, high genetic diversity has often been detected at the centimeter scale. For example, Douhan and Rizzo (2005) found sclerotia formed by three distinct lineages of C. geophilum from a single one-liter soil sample in oak forests in California, USA. Matsuda et al. (2015) studied several widely spaced coastal pine forests in Japan and found that one multi-locus genotype was present on Pinus thunbergii ectomycorrhizal roots in most soil samples (3 cm in diameter and 30 cm in depth). One-third of the samples, however, contained several different genotypes. Obase et al. (2016a) classified isolates of C. geophilum obtained from mixed pine-oak forests in Florida and Georgia (USA) into genotypes based on GAPDH sequences. They found that 75% of soil samples (7 7 10 cm) contained more than one genotype. Half of the samples included 2–3 genotypes but in the remaining 25% of the soil cores up to nine different genotypes were found to co-exist in these small samples. Mechanisms that are involved in structuring such high genetic diversity at a fine spatial scale remained unclear. However, our recent research has explored one possible factor. Several previous studies that have focused on genetic diversity of C. geophilum have used either sclerotia (Portugal et al. 2001; Douhan and Rizzo 2005; Gonçalves et al. 2007) or ectomycorrhizal roots (Panaccione et al. 2001; Matsuda et al. 2015) for molecular analyses. Although either approach is valid as a way of detecting diversity within C. geophilum, it is possible that unique pools of genotypes are present in sclerotia versus ectomycorrhizal roots due to different turnover rates. Sclerotia are excellent resting structures that can remain viable in soil for a long period of time (several years; Trappe 1962), while ectomycorrhizal roots likely remain active for much less time (Fernandez et al. 2013). Obase et al. (unpublished) compared genotypic diversity in C. geophilum isolates from sclerotia and from ectomycorrhizas that were collected in the same 7 7 10 cm soil samples (see Obase et al. 2016a for sampling details). They found that many genotypes were unique to sclerotia or ectomycorrhizas and >50% of genotypes were unique to only one of the sources in most samples. Rarefaction analysis indicates that genotypic diversity was significantly higher in sclerotia than in ectomycorrhizas. This finding suggests that the pool of genotypes that are actively 308 K. Obase et al. growing on ectomycorrhizal roots are a more limited subset of the local genotypic diversity than the genotypes found as sclerotia. Furthermore, this suggests that different life forms (e.g. sclerotia versus ectomycorrhizas) play different roles in structuring the high genetic diversity of C. geophilum. The results also indicate that (1) sampling both sclerotia and ectomycorrhizas is optimal to maximize the detec- tion of genetic diversity in C. geophilum at a fine spatial scale and that (2) intensive sampling effort is probably required in many habitats to adequately assess the genetic diversity of C. geophilum due to the complexity at a fine spatial scale (Obase et al. 2016a).

14.5 Patterns in C. geophilum at Larger Geographic Scales

Many ectomycorrhizal fungi produce sporocarps and disperse large numbers of spores via wind or mammalian mycophagy to colonize new habitats and increase genetic diversity at the landscape scale. Both spore dispersal and vegetative hyphal growth play important roles for structuring the spatial genetic structure of ectomycorrhizal fungi (e.g., Douhan et al. 2011). In contrast to other ectomycorrhizal fungi, C. geophilum has been considered a putatively asexual fungus, and no spores have ever been convincingly discovered. It has been suggested that the vegetative sclerotia and extending hyphae of C. geophilum are the only means of dispersal for this fungus. In theory, these dispersal mechanisms should be less efficient than the large numbers of microscopic spores that are produced by most fungi and distributed by wind, water, or animals (e.g., Maser and Maser 1987). Because there are no other known anamorphic ectomycorrhizal fungi for which spatial genetic structures have been studied, it is difficult to predict the spatial genetic structure that should be hypothesized for C. geophilum. How- ever, if sclerotia and hyphal growth are truly the only dispersal mechanisms for C. geophilum then we might expect highly localized populations with evidence of limited gene flow. However, many recent studies found evidence of cryptic recombination in organisms that were previously considered to be asexual (Kück and Poggeler€ 2009). For example, the human-pathogenic fungus Aspergillus fumigatus was considered to be asexual but were later shown to undergo cryptic recombination (O’Gorman et al. 2009). Several authors have recently suggested that C. geophilum may also undergo cryptic recombination (Douhan et al. 2007a; Bourne et al. 2014; Matsuda et al. 2015; Peter et al. 2016). If present, cryptic recombination would certainly alter the spatial genetic structure in C. geophilum populations (as compared to a completely asexual lifecycle based only on clonal propagation). Only a handful of studies have conducted detailed population-level research in C. geophilum. Wu et al. (2005) investigated the genetic structure of four C. geophilum populations in Salix reinii patches across several kilometers in an early successional volcanic desert on Mount Fuji in Japan using five microsatellite markers. They found that genotypic assemblages of C. geophilum were spatially 14 Progress and Challenges in Understanding the Biology, Diversity, and... 309 heterogeneous. Two of the populations harbored many common genotypes whereas the remaining populations did not share any overlapping genotypes. Wu et al. (2005) inferred that frequent avalanches may transfer sclerotia of C. geophilum with scoria to lower positions on the slope and therefore contribute to the shared genotypes between some patches. They also found that two geographically close populations of C. geophilum did not harbor common genotypes and they suggested that a small valley between the populations could act as a barrier for gene flow. Next, they suggested that recombination in the population was absent or rare due to the fact that there were no ‘intermediate’ genotypes between the two distinct groups of genotypes in each population. These results indicate that spatial distance and other physical barriers may contribute to spatial genetic structuring of C. geophilum at the spatial scale of kilometers. One problem with the Wu et al. (2005) study is that they did not account for the fact that C. geophilum is a species complex and it is therefore likely that they sampled a mixed population that included several cryptic species. Accordingly, it is difficult to fully interpret the results of this study and to determine whether the results might change if the cryptic phylogenetic species of C. geophilum had been recognized. Matsuda et al. (2015) studied the spatial population structure of C. geophilum but they accounted for cryptic species by selecting one dominant lineage using GAPDH barcoding followed by phylogenetic analysis. They found significant genetic variation and no significant spatial autocorrelation within each stand of P. thunbergii coastal forests (1–5 ha). In most cases, although identical genotypes were not detected from adjacent soil samples within each stand, they were infre- quently detected from samples that were 10–50 m apart (and in some cases even >100 m apart), indicating that genet size may be small or genets may be spatially fragmented. It is possible that the high genetic diversity of C. geophilum is maintained by cryptic recombination processes at the landscape scale. Indeed, linkage disequilibrium tests favored recombination as a more likely explanation for the genetic variation rather than clonal reproduction. Next, genetic distance among the populations was weak but significantly correlated with geographic distance (17–1364 km), suggesting a pattern of isolation by distance (Fig. 14.3). The result indicates that unknown migration events might influence spatial distribution and genetic structure of C. geophilum in coastal pine forests at the regional scale. The study by Matsuda et al. (2015) therefore suggests that the spatial genetic structure of C. geophilum is actually somewhat similar to the genetic structure of other ectomycorrhizal fungi that disperse predominantly via spores and less via mycelial growth. Even though C. geophilum is found in forests on many continents, no population genetic studies have been conducted at the global scale. However, Obase et al. (2016a) inferred some broader patterns in the genetic diversity of C. geophilum at the continental scale. The combination of ITS and GAPDH resolved several well- supported phylogenetic clades which included isolates from different geographical regions in North America and several European countries. This suggests that some C. geophilum lineages have dispersed widely within and between continents or that cryptic long-distance dispersal is ongoing via some unknown method. On the other 310 K. Obase et al.

Fig. 14.3 (a) Locations of Cenococcum geophilum populations in several coastal pine forests in Japan. (b) Relationship between genetic differentiation among the populations of C. geophilum (y- axis) and geographic distance (x-axis). The ﬁgure was modiﬁed from Matsuda et al. (2015) hand, even though isolates from different geographical regions (USA, Japan and Europe) were clustered together in the phylogenetic tree, another analytical tool (PTP; Zhang et al. 2013) further delimited them into several taxa per major lineage. Based on this stricter, narrower approach, few of the C. geophilum taxa harbored samples from different geographical regions or continents. Also, several isolates from the same study sites (e.g. same forest stands) were often delimited into several species and isolates from different geographic regions (e.g. California and Florida, USA) were often interspersed between one another in the trees, suggesting that there are no obvious broad biogeographic inferences that can be made without data from more genes and more isolates. Together the available data indicate that the broad phylogenetic approach used by Obase et al. (2016a) to identify major lineages probably underestimates the total number of cryptic taxa. Also, it is possible that there has been both sympatric and allopatric speciation at nearly the same rate among different continents, regions, and sites for each of the phylogenetic lineages thereby helping to partially explain the high cryptic diversity within C. geophilum.

14.6 Future Directions

A series of population genetic studies suggest that cryptic recombination, geographic distance, and physical barriers may structure the spatial genetic patterns in C. geophilum at the forest stand and regional scales, similar to what has been observed for other ectomycorrhizal fungi. However, we still have limited knowledge of the ecology of C. geophilum, particularly when it comes to reproduction and dispersal. The presence of cryptic species that can only be identified via genetic screening (e.g. DNA sequencing or other similar molecular approaches) makes it even more challenging to understand the broad biogeographical patterns in this 14 Progress and Challenges in Understanding the Biology, Diversity, and... 311 group of fungi. Unfortunately, we also lack information on how environmental factors influence the spatial genetic structure of different lineages in C. geophilum. We know that many other soil fungi are strongly influenced by the distance from the equator and mean annual precipitation (Tedersoo et al. 2014) whereas ectomycorrhizal fungi are also clearly affected by the diversity and composition of the host plants. We assume that because C. geophilum is found on the roots of a wide range of ectomycorrhizal host plants in nature and that individual isolates can form ectomycorrhizas on the roots of phylogenetically distinct plants (e.g., pines and oaks), the host does not have a strong influence on the population structure. However, some evidence of host specificity has been documented by inoculation tests using several C. geophilum isolates on different host plants (Antibus et al. 1981). Next, although Gonçalves et al. (2007) indicated that soil properties did not influence genotypic differentiation among serpentine sites, we still cannot rule out the possibility that unique environments may select given genotypes (Branco et al. 2015) and therefore contribute to structuring of C. geophilum populations. Although C. geophilum is challenging to study due to the unknown aspects of the life cycle, this fungal group has the potential to be a model system for studying ectomycorrhizal fungi because it is so widespread in many habitats from tundra to rain forests. Since C. geophilum is culturable and can be found in such widely varying forest types this group would be ideal for studying how genotypic diversity and population genetic patterns are influenced by various kinds of abiotic and biotic factors. For better understanding of spatial genetic structure over different geographical scales, the meta-analysis of spatial genetic patterns in C. geophilum is needed. In the future, it could be helpful to establish common sampling schemes in studies across different sites so that results from different studies and across different biomes could be easily compared to one another. Another challenge for studying C. geophilum is that genetic markers that are used to study one cryptic species do not always work well on the other cryptic species so that individual markers have to be developed for each lineage. Selection of samples based on ITS and GAPDH sequences is a useful first step in any molecular pipeline because both markers are easily amplified from cultures, sclerotia, or ectomycorrhizal roots of C. geophilum. Furthermore, the GAPDH locus is phylogenetically informative and has a growing database of identified samples. In the future, it will be best to follow ITS þ GAPDH screening with next generation sequencing (NGS) strategies that have been recently developed for population genetics. For example, RAD-seq (ran- domly amplified DNA sequencing) is a powerful tool that can generate several hundreds to thousands of genetic markers applicable to different samples that contain cryptic species. So far, there have been no population studies that have used NGS sequencing approaches to examine the population biology of C. geophilum. Using this type of high-throughput approach in combination with sampling across several global regions (e.g. in areas that have remained unsampled for diversity of C. geophilum such as central Asia, Oceania, Africa and South America) would certainly provide a new, comprehensive view of the biogeography of C. geophilum. 312 K. Obase et al.

The use of powerful NGS tools may also potentially provide insights into the unknown ecology of C. geophilum. The first matter of concern is the possibility of recombination among individuals of C. geophilum. Recent studies of genome sequencing found genes related to recombination, i.e., mating genes, in several ascomycetous fungi for which the mating systems were not previously understood. These studies have showed for example that Tuber spp. are heterothallic (Rubini et al. 2011; Belfiori et al. 2013) and also have identified sexual recombination in fungi that were previously considered to only reproduce asexually, such as Asper- gillus (Poggeler€ 2002) and Ulocladium spp. (Geng et al. 2014). The recent study found that one isolate of C. geophilum had one mating-type gene (MAT1-1-1) that was intact and conserved with close relative Glonium species which form fruiting bodies. The presence of genes involving recombination and forming fruit bodies in the genome of C. geophilum indicates the possibility that C. geophilum retains the ability to have sexual recombination like its close relatives in the genus Glonium (Peter et al. 2016). If the presence of other mating type genes (i.e., MAT1-2-1) is found in other isolates of C. geophilum, then spatial patterns of C. geophilum individuals in relation with the mating types may provide insights about how recombination occurs spatially in forests. This type of data would provide critical information about how populations of C. geophilum are structured and whether mating is common, rare, or truly absent in this group of fungi.

Acknowledgements This study was supported by Grant-in-Aid for JSPS Postdoctoral Fellow for Research Abroad (to K. Obase) with additional funding from the University of Florida Institute for Food and Agricultural Sciences (IFAS) (to M. E. Smith). Support for the participation of M.E. Smith was also due in part to US National Science Foundation grant DEB-1354802. We thank the Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida for performing DNA sequencing and the University of Florida’s Austin Cary Memorial forest and Ordway Swisher Biological Station for providing study sites. We appreciate the collaborators at the Smith Mycology Lab at the University of Florida for their assistance in collecting samples.

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